Human replication protein A (RPA), also human single-stranded DNA-binding protein (HSSB), is a single-stranded DNA-binding protein that also interacts and regulates the function of a number of other cellular proteins. This heterotrimeric protein is composed of three subunits, the 70 kDa subunit (RPA70 or p70), p34 (RPA34) and p11 (RPA11) encoded by genes located on different chromosomes (sometimes referred to as RPA1, RPA2 and RPA3 genes, respectively). Each of the three subunits of RPA has been shown to be essential for DNA replication, homologous recombination and nucleotide excision repair in vitro (He, Z., et al., Nature, 1995, 374, 566-569), and disruption of any of the three subunits in yeast is lethal (Brill, S. J. and Stillman, B., Genes and Development, 1991, 5, 1589-1600).
RPA70 binds to single-stranded DNA and certain double-stranded sequences with high affinity (Lao, Y., et al., Biochemistry, 1999, 38, 3974-3984) and possesses unwindase activity. RPA also binds cisplatin (Patrick, S. M., and Turchi, J. J., Biochemistry, 1998, 37, 8808-8815) and UV-damaged DNA. In addition to its DNA binding, RPA interacts with various proteins involved in DNA replication, repair, recombination, transcription and cell regulation. RPA70 interacts with DNA polymerase .alpha., during initiation of replication and elongation, and DNA polymerase .delta. (Longhese, M. P., et al., Mol. Cell. Biol., 1994, 14, 7884-7890). Both RPA70 and RPA34 interact with the Xeroderma Pigmentosum group A complementing protein (XPA) on damaged DNA recruiting endonucleases involved in DNA repair (Stigger, E., et al., J. Biol. Chem., 1998, 273, 9337-9343). RPA also interacts with transcriptional activators including the tumor suppressor gene, p53 (Miller, S. D., et al., Mol. Cell. Biol., 1997, 17, 2194-2201).
The function of the RPA70 subunit has been studied primarily with mutant RPA1 genes (Longhese, M. P., et al., Mol. Cell. Biol., 1994, 14, 7884-7890) and deletion analysis.
RPA70 is believed to be an attractive target for cancer therapeutics. In a preferred embodiment, such a therapy can take advantage of natural genetic variation within the genome in combination with loss of heterozygosity (LOH) in cancer cells. This approach is based on allele-specific targeting and is described in WO 98/41648, herein incorporated by reference in its entirety.
It is estimated that natural genetic variation occurs in approximately one nucleotide in 300 throughout the genome (Cooper, D. N., et al., Human Genetics, 1985, 69, 201-205). Because of the large number of polymorphisms or sequence variances found in the human genome, most individuals are heterozygous for one or more sequence variances in genes of normal tissues, including many genes that are essential for cell survival. LOH reduces many of these genes to hemizygosity in cancer cells, eliminating heterozygosity, and creating a large number of absolute genetic differences between tumor and normal cells (Cavenee, W. K., et al., Mutat. Res., 1991, 247, 199-202; Schwechheimer, K. and Cavenee, W. K., Clin. Investig., 1993, 71, 488-502).
An early event in the clonal evolution of cancers is the loss of large chromosomal regions or even whole chromosomes (Lengauer, C., et al., Nature, 1998, 396, 643-649). Presumably, these losses are driven, in part, by positive selection for cells in which LOH leads to the loss of tumor suppressor functions. LOH in certain cancers can involve more than 20% of the total genome (Lengauer, C., et al., Nature, 1998, 396, 643-649) and it is evident that thousands of genes are also lost from cancer cells due to LOH. Based on current estimates of human gene number this suggests that 15,000 to 20,000 genes, that are not tumor suppressor genes, are also reduced to hemizygosity in cancer cells by LOH. Among these genes are many that are essential for cell survival. The RPA70 gene has been mapped to chromosome 17p13.3 in close proximity to the tumor suppressor gene p53 at position 17p13.1 (Umbricht, C. B., et al., Genomics, 1993, 20, 249-257). This segment of the genome is affected by LOH in many common epithelial cancers (Rodriguez, E., et al., Cancer Res., 1994, 54, 3398-3406). Thus, RPA70 represents an attractive target for allele-specific therapy.
By exploiting the absolute genetic differences in RPA70 genes between cancer cells and normal cells that arise as a consequence of normal genetic variation and LOH, RPA70 can be an effective target for cancer therapy. Inhibitors, especially antisense compounds, are identified that inactivate one or more variant forms of the target gene, but not the normal form that is present in the general population. Inhibitors specific for the remaining allele expressed in the cancer cells, when administered to patients, would be selectively toxic to the cancer cells. Normal cells and tissues, which express both the sensitive and insensitive alleles, would escape significant toxicity.
There remains a long-felt need for improved compositions and methods for inhibiting RPA 70 kDa subunit gene expression.